1. Field of the Invention
The present invention concerns a method of detecting hemolysis in a whole-blood sample, a method of determining an elevation in the potassium ion concentration of a whole-blood sample, an apparatus for detecting hemolysis in a fluid sample, an apparatus for detecting hemolysis in a whole-blood sample, an apparatus for determining an elevation in the potassium ion concentration of a whole-blood sample, and a single-use cartridge containing a plurality of microfabricated biosensors further containing a hemolysis detection unit.
2. Discussion of the Background
A number of analytes in biological fluids are present in amounts which differ dramatically from their concentration in red blood cells. For example, the potassium concentration inside intact red blood cells is usually about 150 mM and the potassium concentration in a plasma fraction of a normal patient is usually about 4.0 mM.
Procedures for manipulating cells or biological fluid samples containing cells can sometimes result in rupture of the cells, a phenomenon known in the art as "lysis." This phenomenon is particularly common for red blood cells. When intact red blood cells are physically damaged and break open, the phenomenon is known as "hemolysis." For example, hemolysis may occur when a blood sample is drawn from a patient. Hemolysis results in mixing the contents of the red blood cells, which contain relatively high concentrations of potassium and hemoglobin (among other analytes), with the plasma fraction of the blood sample.
Hemolysis is common in blood samples, and may occur when blood contacts foreign surfaces. Hemolysis may be unavoidable, especially during cardiac surgery or cardiac bypass, when there is transfusion or extracorporeal circulation. Hemolysis may also result from improper drawing and handling of biological fluid specimens, and can even occur during centrifugation and separation procedures. Therefore, clinical chemists must know whether hemolysis affects the analyses ordered by clinicians. Many laboratories reject all hemolyzed specimens without considering whether this approach is justified.
In clinical medicine, for example, the potassium concentration in a whole-blood sample is determined by measuring the potassium concentration in the plasma fraction of the whole-blood sample. In view of the fact that the potassium concentration inside red blood cells can be from 25 to 75 times higher than the potassium concentration in blood plasma, hemolysis of only a few red blood cells will result in an artificial elevation of the concentration of potassium in the plasma fraction.
In the medical arts, generally only the potassium concentration in the plasma fraction is important for determining the electrolyte balance of a patient. The potassium concentration in the plasma fraction is used to determine, for example, the sodium/potassium balance, an important indicator of proper nerve conduction, and other fundamental conditions related to acute care known to those in the medical arts. Consequently, hemolysis during sample draw can result in an over-estimate of the true plasma potassium concentration. This can be particularly serious if a patient actually has a low plasma potassium concentration, requiring urgent treatment. In this scenario, a small number of hemolyzed red blood cells can elevate the plasma potassium concentration into the normal range, resulting in no treatment where urgent treatment is required.
In addition to potassium, measurements of the concentration of lactate dehydrogenase and acid phosphatase are significantly affected by hemolysis, and an increase in cholesterol levels may be observed when there is severe hemolysis.
Erythrocytes (red blood cells) contain about 160 times more lactate dehydrogenase, 68 times more acid phosphatase, 40 times more aspartate aminotransferase, and 6.7 times more alanine aminotransferase as does blood plasma (Caraway, Chemical and diagnostic specificity of laboratory tests. Am. J. Clin. Pathol., 1961; 37:445-64). Lactate dehydrogenase and acid phosphatase are the clinically most important enzymes which show significant increases because of hemolysis. Prostatic acid phosphatase (tartrate-inhibited acid phosphatase) is also affected by hemolysis. It is believed that tartrate may not inhibit the increase in enzyme activity resulting from hemolysis (see Yucel et al, Effect on in vitro hemolysis on 25 common biochemical tests. Clinical Chemistry, 1993, 38:575-577).
For this reason, phlebotomists and other medical personnel who are responsible for drawing blood samples are taught techniques that minimize hemolysis. For example, when obtaining a sample by means of a finger or heal-stick, squeezing the site to promote blood flow is known to cause hemolysis, and should be avoided.
The standard clinical chemistry laboratory method for determining if a blood sample has significant hemolysis is to spin the sample in a centrifuge to separate the plasma fraction from the red blood cells, then visually examine the plasma fraction. Hemolysis results in the plasma fraction being contaminated with hemoglobin, which gives an obvious red color to the otherwise yellow plasma. In a typical clinical chemical laboratory, virtually all blood tests performed are based on plasma measurements. Accordingly, centrifuging the blood sample is a satisfactory procedure to determine hemolysis in the clinical laboratory.
As a result, if a test ordered by a physician is known to be subject to interference due to hemolysis (e.g. potassium, lactate dehydrogenase, acid phosphatase; see Yucel et al) and the result is abnormal, the color of the plasma sample will be checked by a laboratory technician. If the plasma fraction shows evidence of hemolysis, a fresh blood sample will generally be obtained.
Although the effects of hemolysis are method-dependent (Frank et al, Effect of in vitro hemolysis on chemical values for serum. Clin. Chem., 1978; 24:1966-1970), even when samples are only slightly hemolyzed, results for lactate dehydrogenase, acid phosphatase, prostatic phosphatase, and potassium analyses must be rejected.
The concentration of free hemoglobin (Hb) in serum can be used as a measure of hemolysis (Yucel et al). For example, serum or blood plasma shows visual evidence of hemolysis when the hemoglobin concentration exceeds 20 mg/dL or is greater than 3.1 .mu.mol/L (Caraway, supra; Sonntag et al, Haemolysis as an interference factor in clinical chemistry. J. Clin. Chem. Clin. Biochem., 1986; 24:127-139). Hemolysis has little effect on constituents that are present at lower concentrations in erythrocytes than in plasma, but a marked effect may be observed for constituents that are present at a higher concentration in erythrocytes than in plasma (Sonntag et al, supra; Frank et al, supra; Brydon et al, The effect of haemolysis on the determination of plasma constituents. Clin. Chem. Acta, 1972; 41:435-438). Hb may also interfere in the colorimetric determination of constituents when certain chromogenic reagents susceptible to oxidation by Hb are used to provide a visual indication of the presence and/or concentration of the constituents.
In addition to the analytes described above (potassium, lactate dehydrogenase, cholesterol, prostatic phosphatase, aspartate aminotransferase, and alanine aminotransferase), concentrations or activities of aldolase, total acid phosphatase, isocitrate hydrogenase, magnesium and phosphate are also increased by hemolysis in biological fluid samples.
For example, when the biological fluid is whole-blood, the inorganic phosphate concentration in the corresponding plasma fraction increases rapidly as the organic esters in the cells are hydrolyzed. Aspartate aminotransferase activity is increased by 2% for each 10 mg hemoglobin per dL of biological fluid sample (Hb/dL). When colorimetric procedures without extraction are used, 10 mg Hb/dL will raise the apparent cholesterol concentration by 5 mg/dL. Ten mg of hemoglobin per dL of biological fluid sample will increase serum lactate dehydrogenase by about 10%, and serum potassium by about 0.6% (see Tietz, "Textbook of Clinical Chemistry," W. G. Saunders and Co. (1986), page 488).
A method and test strip for determining the presence of hemoglobin in urine is known (HEMASTIX.TM., manufactured by Miles Ames, Elkart, Ind.). However, the HEMASTIX.TM. test is only semi-quantitative. Further, there is no need to discriminate between red blood cells and Hb in the HEMASTIX.TM. test, and as a result, red blood cells need not be separated prior to use of HEMASTIX.TM. strips. In addition, urine usually has an acidic pH, whereas blood serum or plasma has a pH of 7.4. Thus, the method and test strip used to determine the presence of Hb in urine falls short of the need for a method and device for determining hemolysis in whole blood.
Further, methods of separating plasma or serum from whole-blood using a material which separates blood plasma or serum from erythrocytes and leukocytes or from whole-blood are also known (U.S. Pat. Nos. 4,477,575 and 4,816,224). However, these methods and apparatuses have not been used to determine the presence of hemoglobin or an elevation in the concentration of a blood analyte due to hemolysis of red blood cells.
Currently, there is no practical method for determining whether a blood sample is hemolyzed when tested with any analytical system which:
(i) operates on whole-blood, and PA1 (ii) is used at the patient's bedside, where a centrifuge is not available. PA1 (a) contacting a whole-blood sample comprising intact red blood cells with a dry separation material, said material having physical characteristics effective to separate a fraction, which contains extracellular hemoglobin that may be present in said sample, from the intact red blood cells; PA1 (b) allowing said sample to remain in contact with said material for a period of time sufficient to effect said separation; and PA1 (c) detecting the presence of extracellular hemoglobin in said fraction, PA1 and in which the apparatus comprises: PA1 (a) a dry separation material to which a fluid sample may be applied, said material having physical characteristics effective to separate a fraction of said sample, which fraction would contain a representative concentration of any extracellular hemoglobin that may be present in said sample; and PA1 (b) a detection means for ascertaining the concentration of said extracellular hemoglobin in said fraction.
The present invention seeks to solve this problem by creating a simple method and device for identifying blood samples in which hemolysis has occurred to a level sufficient to result in erroneous measurements of plasma analyte concentrations or activities.